Evaluation of in vitro antidiabetic and antioxidant activity of leaf extracts of Ecbolium linneanum kurz.: GC-MS and HR-LCMS-based metabolite profiling and an in silico approach

The Ecbolium linneanum Kurz., plant has been used historically in traditional medicine to treat a wide range of ailments including gout, rheumatism, etc. Therefore, we set out to investigate its anti-diabetic and antioxidant effects. This study was executed by assessing its in vitro antidiabetic and antioxidant effects and characterizing the phytochemical composition of the plant material. The antidiabetic action was measured using the α-amylase assay, while antioxidant activity was evaluated using the 2,2-Diphenyl-1-picrylhydrazyl) (DPPH), ferric ion reducing antioxidant power, and hydrogen peroxide assays. The compounds were also put to in-silico tests for drug-likeness, absorption, distribution, metabolism, and excretion profile, and binding interactions with α-amylase. In this study, the methanolic leaf extract significantly inhibited α-amylase activity (IC 50 – 684.94 g/ml). An impressive level of antioxidant activity (87.78% radical scavenging) was measured with the DPPH test. About 57 phytochemicals were discovered in the methanolic leaf extract employing gas chromatography-mass spectroscopy and high-resolution liquid chromatography-mass spectrometry analyses. About 28 compounds were found promising in oral bioavailability and drug-likeness properties in in-silico research. A few compounds like rhodotoxin A, pyrophosphite a, and 23-acetoxysoladulcidine demonstrated a high-binding affinity for α-amylase. The findings of this study provide preliminary evidence that leaf extracts of E. linneanum may possess anti-diabetic and antioxidant activities. This plant is an excellent source to instigate in researching new natural candidates for antidiabetic therapy.


INTRODUCTION
Diabetes mellitus (DM), which is notable for improper lipid, lipoprotein, and glucose metabolism, has emerged as one of the most common illnesses and is on the rise globally.The Centre for Disease Control and Prevention has recently released the 2022 National Diabetes Statistics Report, as per the report estimates more than 130 million adults are living with diabetes or prediabetes in the United States.The prevalence of DM and impaired fasting blood glucose in India was 9.3% and 24.5%, respectively [1].Among those with DM, 45.8% were aware, 36.1% were on treatment and 15.7% had it under control.It is projected that by 2025 the number of cases of diabetes in India would be 69.9 million with a vast majority still undiagnosed.Given that controlling diabetes can be done with a variety of current treatments, such as insulin injections and oral medications like hypoglycemic pills, the majority of them suffer from a variety of drawbacks, such as being costly or coming with side effects like hepatic toxicity, weight gain, hypoglycemia, gastrointestinal (GI) disturbances, and GI hemorrhage.Because of their accessibility, efficacy, and historical, cultural, and religious preferences, medicinal herbs continue to be a widely used alternative medicine.According to estimates from the WHO, 65%-80% of the world's population lives in underdeveloped nations due to budgetary limitations, Evaluation of in vitro antidiabetic and antioxidant activity of leaf extracts of Ecbolium linneanum kurz.: GC-MS and HR-LCMSbased metabolite profiling and an in silico approach Srilakshmi Nallapaty 1 , Narender Malothu 1* , Sathish Kumar Konidala 2 , Anka Rao Areti 1 identified by gas chromatography-mass spectroscopy (GC-MS) and high-resolution liquid chromatography-mass spectrometry (HR-LCMS) investigations and were further analyzed virtually for binding interactions with α-amylase using the Protein Data Bank (PDB) crystal structure (1b2y).

Plant material
In the first week of September 2018, E. linneanum leaves were acquired as raw material from the wastelands of Guntur, Andhra Pradesh, India.The collected leaves were recognized by Dr. Madhav Shetty, a professor in the Department of Botany at Sri Venkateshwara University in Tirupati, Andhra Pradesh, India.The leaves were scrubbed until there was no longer any sign of foreign matter in distilled water, detergent, and tap water.To air dry, the leaves were kept at room temperature in the shade.The dried leaves were freed from the moisture and processed into a powder using a sterile electrical blender.The powdered substance was then kept in an airtight container for later use [8].

Extraction
A Soxhlet apparatus was used to get the crude leaf extracts of petroleum ether (PEEL), ethyl acetate (EAEL), and methanolic (MEEL) from 180 g of coarse powder throughout 72 hours.By utilizing a rotary evaporator, the crude filtrate was concentrated.The resultant extracts were kept in a refrigerator at 4ºC for later use [8].

Phytochemical analysis
To determine the presence of a variety of secondary metabolites in leaf extracts qualitative phytochemical testing was conducted.The earlier investigations presented that the MEEL was comprised of rich amounts phytochemicals, such as alkaloids, glycosides, phenols, and flavonoids [8].

Total phenolic content (TPC)
The FC colorimetric method [9] was used to assess the TPC of E. linneanum leaf extracts.About 100 µl of the diluted extracts, 500 µl of the FC reagent, and 400 µl of 7.5% (w/v) saturated aq.Na 2 CO 3 were combined thoroughly.Then, it was homogenized and incubated for 30 minutes at 40ºC.After incubation, each sample's absorbance was measured at 765 nm using methanol as the blank.The calibration curve approach was used to ascertain the TPC using gallic acid as the standard.Each test was run in triplicate, and the results were expressed as gallic acid equivalents (GAE) in mg per g of dry extract (mg GAE/g) [9].a lack of access to modern medications, and relies primarily on naturally occurring items for their initial medical care.The global market for medicinal plants expanded from US $23 billion in 2002 to more than US $83 billion in 2008.Only 10% of the world's (2.5 million) species have had the therapeutic potential of their plants and herbs examined since World War I, irrespective of the fact that there has been extensive research done on the problem since that time.This implies that there are still many effective medications that have not yet been explored.
As oxygen is crucial to the survival of life on earth, a small amount of oxygen is converted to numerous free radicals as it is utilized in various living processes.In addition to the abovementioned pollutants, endogenous ones generated by metabolic processes are also exposed, as are various chemicals and the climate.Reactive oxygen species (ROS) such as superoxide anion (O2•-), hydroxyl (HO•), and peroxyl (ROO•) radicals, as well as reactive nitrogen species (RNS) such as peroxynitrite anion (ONOO-) and nitric oxide (NO•) radical, are present in the exposed oxidants; overall harmful effects occur when these species attack human cells and tissues, leading to cancer [2].Natural antioxidants in high quantities are frequently found in plants.Antioxidants can stop a chain reaction by scavenging free radicals and preventing oxidation processes [3].The phytochemicals found in plants, such as phenols, flavanols, carotenoids, and vitamins C and E, can be employed to remove excess radicals from the human body [4].
Ecbolium linneanum, is an indigenous Indian plant that naturally grows along India's eastern coast.Also, it can be found in tropical Asia and Africa.There have been numerous traditional uses for E. linneanum, including the treatment of jaundice, menorrhagia, rheumatism, inflammation, etc. Root juice is used to treat premenstrual colic and as an anti-helminthic [5].The plant is being used for gout, dysuria, and stricture with a leaf decoction.The plant's leaves and roots are used to cure malignancies and used to treat cardiovascular diseases.The extracts from various plant parts, particularly leaf extract, are high in polyphenolic substances such as phenols and flavonoids [6].Plant components are thought to provide a variety of beneficial health impacts, such as antioxidant properties, which prevent cell damage from ROS and RNS, which can be free radicals, singlet oxygen, and hydroperoxides.Cell damage caused by free radicals is one of the primary causes of aging and several degenerative disorders of aging, such as stress, cancer, cardiovascular disease, immune system degeneration, DM and inflammation, etc. Free radicals can be scavenged or quenched by the body's enzymes, such as glutathione peroxidase and superoxide dismutase, to defend the body from negative impacts.These enzymes might be assisted in doing so by phytochemicals that exist in plants.Numerous assays, such as the DPPH, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid, ferric ion reducing antioxidant power (FRAP), and CUPRAC tests, have been used to evaluate the antioxidant capabilities of plant extracts.These methods have shown varying results among the examined plants and laboratories.
The leaves of E. linneanum have been demonstrated to be a possible hypoglycemic source in an earlier investigation [7].Therefore, we broadened our study to include in vitro assays on target enzyme activity, such as α-amylase, to assess the antidiabetic potentials of leaf extracts.In vitro, testing was used to ascertain the extracts' anti-oxidant properties.Phytocompounds

Total flavonoid content (TFC)
TFC of E. linneanum leaf extracts was assessed by the aluminum chloride (AlCl 3 ) colorimetric technique [10].In this method, 125 µl of sodium nitrite solution (5%), 250 µl of each solvent extract, and 1,250 µl of distilled water were combined.The contents were then let to stand for 6 minutes at room temperature.150 µl of 10% AlCl 3 solution was then added, and the mixture was left to stand for another 6 minutes.Following that, 275 µl of distilled water was used to dissolve 500 µl of sodium hydroxide solution (4%).The mixture was completely combined and left to stand at room temperature for 30 minutes.At 510 nm, the absorbance was measured in comparison to a reagent blank that contained methanol.The measurement of total antioxidant capacity was performed using quercetin as a reference (QE) per mg of dry extract (µg QE/mg) [11].

In vitro antidiabetic activity
The in vitro antidiabetic activity was assessed by performing α-amylase inhibitory assay.In which the sample solution (100 µg/ml) of all the solvent extracts was made by using phosphate buffer (pH-6.8).500 µl of test solution was added with α-amylase (0.5 mg/ml) solution.The resulting solution was incubated for 10 minutes at 25ºC [12].Further procedure was followed to estimate the inhibitory effect on enzyme activity taking acarbose as a reference standard.The inhibitory effect on the α-amylase activity of solvent extracts was statistically expressed in terms of their IC 50 values [13].

Antioxidant activity
The solvent extracts were subjected to screen for their possible antioxidant capabilities by DPPH, H 2 O 2, and FRAP methods.

DPPH free radical-scavenging assay
The free radical scavenging assay of the extracts (PEEL, EAEL, and MEEL) utilizes the stable free radical of DPPH according to Yeo and Shahidi [14] DPPH methanolic solution (1 ml, 0.2 mM) was added to the extract solution (1 ml, 2.5-100 µg/ml).The mixture was vortexed thoroughly and incubated for 30 minutes at room temperature.The absorbance was measured at 517 nm with methanol as a blank.ABA as a positive control, the DPPH activity was measured by using the below equation: Radical scavenging activity (% inhibition) = (Abs.blank-Abs.sample)/Abs.blank × 100 IC 50 value of solvent extract was calculated from the regression line [14].

H 2 O 2 radical scavenging assay
The H 2 O 2 scavenging assay was carried out by following the standard procedure described by Ruch et al. [15].A solution of H 2 O 2 (43 mM) was prepared in phosphate buffer (0.1 M, pH-7.4).The three extracts (PEEL, EAEL, and MEEL) at different concentrations (1 ml, 2.5-100 µg/ml) in 3.4 ml phosphate buffer were added to 0.6 ml of H 2 O 2 solution (43 mM).The absorbance of the mixture was measured at 230 nm [16].
H 2 O 2 scavenging activity (% inhibition) = (Abs.blank-Abs.sample)/Abs.blank × 100 The IC 50 value of solvent extracts was measured from the regression line of the % of remaining H 2 O 2 radical with the sample concentration.

FRAP assay
The method described by Gohari et al. [17] was used to measure the reducing power of the PEEL, EAEL, and MEEL. 1 ml of test sample solution at various concentrations (2.5-100 µg/ml) was combined with 2.5 ml of potassium ferricyanide (1% w/v), 2.5 ml of phosphate buffer (0.2 M, pH-6.6) and 2.5 ml of 10% trichloroacetic acid, which was then incubated for 20 minutes at 50ºC.After the mixture was centrifuged for 10 minutes at 3,000 rpm, 2.5 ml of the upper layer was removed and combined with 2.5 ml of distilled water and 0.5 ml of FeCl 3 (0.1%).The calibration curve approach was used to determine the % inhibition using the standard ABA (10 g/ml).A spectrophotometer was used to measure each absorbance in triplicate at 700 nm [17].
Ferric reducing power (% inhibition) = (Abs.blank-Abs.sample)/Abs.blank × 100 The IC 50 of solvent extracts was analyzed from the regression line of the % of remaining ferric ion radical at the test concentration of extract.

GC-MS and HR-LCMS study
Conventional methods for identifying bioactive phytoconstituents require a variety of procedures, including extraction, chromatographic separation, and spectroscopic characterization.Nevertheless, despite substantial time and effort, most researchers only attempt to characterize a small number of known phytochemicals due to the lack of appropriate phytochemical standards.To identify and simplify efforts to understand their action on the target, high throughput, and high-resolution approaches must be used to reveal the complex chemistry of bioactive crude extracts.Extensive research has been conducted to better understand the medicinal applications of the Ecbolium genus.However, despite its usage as a replacement constituent for E. linneanum, efforts toward knowing the chemistry of the plant have remained significantly low.The preliminary phytochemical study confirmed that the MEEL comprised diverse classes of phytoconstituents like alkaloids, flavonoids, phenols, saponins, etc. Besides, it displayed significant activity in in vitro and in vivo studies.Thus, as per the findings of qualitative phytochemical tests and biological efficacies, the MEEL was subjected to GC-MS and HR-LC-electrospray ionization (ESI)-MS/MS analysis to determine the presence of diverse phytochemicals [18].

GC-MS analysis
All the probable phytochemicals that are separated from the sample will be detected as a spectral emissary by this approach.After injecting the sample into the GC-MS device's port, the sample is vaporized and then separated using an analyzer.Each component is generating a clean, identifiable peak, which was recorded digitally on a graph.The analysis was performed at CSIR-Indian Institute of Chemical Technology, Habsiguda, Hyderabad.The data was recorded on a combined gas chromatogram system (Agilent GC-MS5977B) and mass spectrometer, fitted with an HP-5 MS fused silica column (5% phenyl methyl siloxane 30.0 m × 250 μM, film thickness 0.25 μM), interfaced with 5675C Inert MSD with Triple-Axis detector.

HR-LCMS analysis
In HR-LCMS analysis, the phytoconstituents were profiled based on the retention time (Rt), m/z values, NIST library hits, and metabolite class.The metabolite analysis in MEEL was performed by HR-LCMS, UHPLC-PDA detector-ESI-QTOP-MS (Agilent Technologies, USA).With the aid of mass spectra and distinctive mass fragmentation patterns, compounds were discovered.For the chromatographic separation, Hypersil GOLD C18 (2.1 × 100 mm 3-µ) column was used with a gradient solvent system, (A) water with 0.1% formic acid and (B) 90 % acetonitrile with 10% water + 0.1% formic acid, A 95% B 5% for 1 minute, B 100% for 2-30 minutes, A 95% B 5%, for 30-35 minutes at 0.3 ml/minute flow rate with pressure maintained at 1,200 bar.The mass spectral data were acquired in both ESI-positive and negative ionization modes.MS was acquired over the m/z range of 100-1,200 at a mass resolution of 22,000 full-width half at maximum [19].

In silico absorption, distribution, metabolism, and excretion (ADME) study
The ADME properties of discovered metabolites in GC-MS and HR-LCMS were assessed using an online tool (SwissADME), to choose the most promising compounds with minimal risk of drug attrition in the later studies.The metabolites with the most reliable ADME properties have been taken into consideration for docking study [20].minimized in 3D structure using ChemDraw software tools and saved as pdb files.The saved .pdbfiles were subjected to energy minimization (force field-off), and then generated conformers (AutoDock pdbqt files) using the open babel tab in PyRx software.The macromolecule (protein pdbqt file) and ligands were chosen using Vina Wizard to do the docking study (Autodock pdbqt files) by drawing a grid box around the area where the co-crystal ligand exhibits iterations with amino acids, the active site of the protein was defined to dock ligands to proteins.The potential compounds with a high binding affinity against the target protein were identified as the ligands with the lowest binding energies.The binding interactions were visualized using the Discovery Studio Visualizer 2021 program.

Docking study
The X-ray crystal structure of the α-amylase (PDB ID: 1b2y) [21] protein co-crystallized with acarbose was retrieved from a protein data bank (rcsb.com/pdbdatabase).The protein was refined by eliminating water molecules, adding polar hydrogen.The co-crystal ligand was extracted using the Discovery studio visualizer 2021 program and saved in pdb format.The protein was checked for any missing amino acid residues, and the Ramachandran plot was used to check for any structural problems.The created protein file in pdb format was converted to pdbqt format using the macromolecule option in the Autodock tool of the PyRx virtual screening application 0.8.The target compounds 2D structures were drawn and their energy was

Antioxidant activity
The antioxidant capabilities of three solvent extracts determined by DPPH, H 2 O 2 , and FRAP assays were presented in the following,

Phytochemical analysis
The initial phytochemical analysis showed that each solvent leaf extract contained a distinctive variety of phytoconstituents.Saponins and tannins were the primary components of all three solvent extracts.However, the ratio of other phytoconstituents varied [7].

TPC and TFC
In Table 1, the amounts of TPC and TFC were shown as mg/g.When compared to the PEEL and EAEL, the MEEL comprised the highest levels of TPC and TFC.The phytochemical content of MEEL was thought to be high, so the plant extract was tested for its ability to combat diabetes and free radical damage [9,11].

S.no
Compound label Rt Mass Formula DB Diff (ppm) Hits (DB)

H 2 O 2 radical scavenging assay
The H 2 O 2 radical scavenging assay was tested at 25-500 µg/ml of three solvent extracts, in which MEEL exhibited the highest (77.22% ± 0.43%) radical scavenging activity at 500 µg/ml.The PEEL and EAEL extracts were moderately potent at 500 µg/ml (Fig. 3).54 Pyropheophorbide a Antioxidant [57] 55 Retapamulin Tricyclic diterpenoid Topical antibiotic [58] 56 Saxitoxin Alkaloid Neurotoxin [59] 57 Sinapoyl malate Coumaric acid Sun screen agent [60] study could provide information on the distribution of various polar to non-polar phytochemicals.ESI positive and negative ion modes were applied to identify all types of small molecules.The representative base peak LC 8chromatogram of MEEL was depicted in Figure 6.Table 3 which summarized the tentative metabolites characterized from MEEL includes their Rt, experimental m/z, mass, proposed metabolites, molecular formula, etc.The HR-LCMS study revealed that the distribution of several (57) bioactive compounds, such as (+/−)-3-[(2-methyl 3-furl) thiol]-2-butanone, 8-hydroxy-2-chlorodibenzofuran, sinapoyl malate, and other phytochemicals.It reveals that there was a considerable number (57) of metabolites present in the MEEL as only a few metabolites have been focused on in the present study.
The present study also revealed the pharmacological richness of E. linneanum wherein 30 bioactive chemical compounds were identified.Examples include steroid compounds like 23-acetoxysoladulcidine and Rhodexin A, flavonoids like 2ʺ, 3ʺ-Di-O-p-coumaroylafzelin, and alkaloids like pheophorbide a and pyropheophorbide a. Out of 57 identified phytoconstituents, 21 bioactive compounds had good oral bioavailability, and certain compounds were promising and

FRAP assay
The FRAP assay was tested at 25-500 µg/ml of three solvent extracts.In this study, the MEEL exhibited 76.56% ± 0.27% of inhibition at 500 µg/ml, which was a similar impact observed in H 2 O 2 radical scavenging assay.The PEEL and EAEL extracts were found less potent at 500 µg/ml (Fig. 4).

HR-LCMS analysis
The m/z values were in the range of 135 to 724 for the majority of separated compounds in MEEL.The HR-LCMS    for the first time on HR-LCMS-based identification of possible phytochemicals, and the profile showed 57 compounds, of which 21 compounds showed significant physiochemical and pharmacokinetic profiles.Among these 2ʺ,3ʺ-Di-O-pcoumaroylafzelin, 23-acetoxysoladulcidine, pheophorbide a, pyro pheophorbide a, and rhodexin, etc.The in silico analysis of the discovered phytochemical compounds predicted that they would have excellent bioavailability and good binding energies.The GC-MS analysis demonstrated the distribution of a few bioactive compounds.According to the literature, one of the compounds γ-Sitosterol has antihyperglycemic activity, and have significant antioxidant activity.Additional research is needed to isolate the more abundant compound in the MEEL by using different methods and to continue further studies for the identified compounds.In future isolation of responsible phytochemicals and their in vivo hypoglycemic and antioxidant assessment may lead to the development of effective novel natural agents.

ABBREVIATIONS
EAEL: Ethyl acetate extract of E .linneanum;FC: Folin ciocalteu; GC-MS: Gas chromatography-mass spectroscopy; HR-LCMS: High-resolution liquid chromatography-mass spectrometry; MEEL; Methanolic extract of E. linneanum; PEEL: Petroleum ether extract of E. linneanum; TPC: Total phenolic content; TFC: Total flavonoid content.supported Lipinski's rule with zero violations in an in silico SwissADME study.Around 28 phytoconstituents listed in Table 4 were selected and performed in silico prediction using acarbose as the standard.

In silico ADME study
Online tools, such as the Swiss ADME free version, were used to evaluate drug-likeness and other pharmacokinetic parameters.A few compounds were found to have strictly adhered to Lipinski rule 5. Table 5 displayed the ADME profile including GI absorption, BBB permeability, and Lipinski rule violations.

In silico docking study
Since α-amylase (PDB ID: 1b2y) is one of the key enzymes that facilitates the release of glucose into the bloodstream, it was selected as a target for in silico docking study.One of the potential approaches to regulating blood sugar involves inhibiting its enzyme activity.Thirty compounds were chosen based on their m/z values, and their properties were predicted in silico with acarbose as the reference compound.The in silico results demonstrated that the 23-acetoxysoladulcidine (−9.6 kcal/mol), rhodexin A (−9.6 kcal/mol), pheophorbide a (−9.5 kcal/mol), and pyropheophorbide a (−9.5 kcal/mol) were significant binding affinities against target (Fig. 7).The binding energies and active-site interactions that are crucial to their binding of ligands screened has been indicated in Tables 6 and 7.
It tends to be of utmost importance to use medicinal herbs as antioxidants and in the treatment of diabetes.This is said to be a result of the safety stigma linked to plants, the high cost, the abundance of adverse effects, and the lack of orthodox medications.The advantages of the secondary metabolites that these plants make to defend themselves from outside invaders are being highlighted in an increasing number of studies.MEEL is abundant in alkaloids, phenols, flavonoids, steroids, and tannins, according to a phytochemical study.For their capacity as antioxidants, tannins, phenols, and flavonoids are widely known.GC-MS and HR-LCMS analysis revealed about 67 phytoconstituents, in which few compounds (Ex.γ-sitosterol) have been found to be anti-diabetic activity [22].Among these 21 compounds met the drug-likeness and other pharmacokinetic parameters when tested in silico.Only 6 of the 28 compounds tested by docking studies had good binding energy.According to the GC-MS and HR-LCMS metabolite profiling results, the volatile compounds γ-sitosterol, and pheophorbide A have been reported to be hypoglycemic.

CONCLUSION
The experimental investigations on PEEL, EAEL, and MEEL suggested that the leaves of the plant were enriched with several biologically active compounds.The MEEL contains a high concentration of flavonoids and phenols, which may account for its high antioxidant activity.In vitro, antidiabetic testing revealed that MEEL significantly inhibited α-amylase activity when compared to acarbose.It has also been reported

Table 2 .
List of phytochemicals identified by GC-MS analysis in MEEL.

Table 4 .
Chemical classes of phytochemicals identified by GC-MS and HR-LCMS analysis.

Table 5 .
ADME profile of identified phytochemicals as per in-silico study.

Table 6 .
Binding energy of phytochemicals in docking study against 1b2y.

Table 7 .
Binding interactions of phytochemicals at the active cite of 1b2y.